Complex Population Structure of Lyme Borreliosis Group Spirochete Borrelia garinii in Subarctic Eurasia

Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete''s samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species'' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles.     

Reservoir Competence of the Vole,Clethrionomys rufocanus bedfordiae,for Borrelia garinii or Borrelia afzelii

In autumn of 1994 and spring of 1995, we examined Borrelia infection among Microtinae voles, Clethrionomys rufocanus bedfordiae, in Hokkaido, Japan. In BSK culturing of the earlobe tissues of 45 C. rufocanus bedfordiae captured, twelve rodents were positive for Borrelia. Eight isolates were used for the polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) analysis. According to the results, these isolates were classified into B. garinii or B. afzelii. It is considered that a common vole, C. rufocanus bedfordiae, plays a significant role in the transmission and maintenance of B. garinii and B. afzelii, similar to the role of Apodemus speciosus mice.     

T Cell Response to Borrelia garinii,Borrelia afzelii,and Borrelia japonica in Various Congenic Mouse Strains

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4⁺8^? and Ia^? T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8⁺ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.     

Genetic variants of Borrelia garinii, a widely spread Eurasian pathogen of ixodic tick borreliosis

As a result of PCR-RFLP analysis and the degree of similarity between the nucleotide sequences analysis of the rrfA-rrlB intergenic spacer DNA of 227 primary isolates of Borrelia garinii and 71 isolates/ amplicons from GenBank database in different regions of Eurasia revealed significant intraspecific heterogeneity among those of Borrelia. It was shown that genospecies B. garinii had within the two genetic subgroups (20047 and NT29) 16 genetic variants, whose geography was likely to be different.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Peromyscus maniculatus, a possible reservoir host of Borrelia garinii from the Gannet Islands off Newfoundland and Labrador

Thirty-five deer mice, Peromyscus maniculatus, were trapped on Gannet Cluster 2 (GC-2), one of a group of islands numbered by convention in the Gannet Island Archipelago, and examined for ectoparasites. One species each of Acari (Ixodes uriae) and Siphonaptera (Orchopeas leucopus) were recovered. Samples of mice favored males to females (3.4∶1). Twenty-nine percent (10) of the mice were free of ectoparasites. Males were more heavily parasitized than females when both parasites were considered. No ticks were recovered from the female mice, while the males that were parasitized carried adult Ixodes uriae. These 2 ectoparasites parasitizing P. maniculatus, which is a known reservoir host for Lyme disease (Borrelia burgdorferi), may carry B. garinii and their presence would have serious implications for the spread of this human pathogen northward in continental North America.     

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy

Abstract This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii , by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii . An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 ( B. afzelii ), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.     

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA^S226A). Although inoculation with either BbHtrA or BbHtrA^S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.     

Demographic and environmental causes of the decline of rural Song Thrushes Turdus philomelos in lowland Britain

We review current knowledge of demographic mechanisms and environmental factors implicated in the population decline of Song Thrushes Turdus philomelos in rural Britain since the mid-1970s, and present new analyses of regional variation in population changes. Increased mortality during the first year of life (from fledging to recruitment) is highlighted as a potential demographic mechanism having driven the population decline, while Song Thrushes in a rapidly declining farmland population were making too few nesting attempts to sustain local numbers. Breeding Song Thrushes are strongly associated with non-cropped habitats such as woodland edge, field boundaries, gardens and scrub; they make substantial use of grassland, but avoid cereals when foraging. Earthworms constitute a key component of Song Thrush diet and the availability of this prey is strongly influenced by moisture levels in surface soils. Several lines of evidence suggest that dry surface soils during summer are deleterious to the productivity and survival of Song Thrushes, and regional variation in the rates of population change in Britain during 1970–86 was negatively correlated with the extent of under-field drainage on farmland (the main function of which is to promote the drying of surface soils). Increasing dryness of agricultural soils and the loss of grassland from eastern arable counties have probably both contributed to the declines of rural Song Thrushes in Britain. Loss of hedgerows and scrub, and the degradation of woodland may also have contributed to population declines but the role of predators remains unclear. Recovery of rural Song Thrush populations requires challenging new policy initiatives that should aim to restore nesting cover (scrub and woodland understorey), grazed grassland in arable-dominated areas and damper soils in summer.     

Relationships of a Novel Lyme Disease Spirochete, Borrelia spielmani sp. nov., with Its Hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Isolation and Characterization of Vibrio carchariae,a Causative Agent of Gastroenteritis in the Groupers,Epinephelus coioides

An outbreak of serious mortality among the cultured groupers Epinephelus coioides, characterized by a swollen intestine containing yellow fluid, occurred in the summer of 1993 in Taiwan. A motile strain EmI82KL was isolated from the intestinal yellow fluid of the moribund groupers with tryptic soy agar supplemented with 2% NaCl and/or thiosulfate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio carchariae and was susceptible to chloramphenicol, doxycycline-HCl, nalidixic acid, oxolinic acid, oxytetracycline, and sulfonamide while resistant to ampicillin and penicillin G. In addition, the strain was neither auto-agglutinating nor hemagglutinating, but it was hemolytic against erythrocytes from sheep, rabbit, tilapia, and grouper. The bacteria could be reisolated from kidney, liver, and the transparent yellow fluid of swollen intestine of moribund groupers after bacterial challenge and re-identified as the same species. The LD₅₀ value was 2.53 × 10⁷ colony forming units/g grouper body weight. Received: 26 December 1996 / Accepted: 20 February 1997     

Mycoplasma Dispar As a Causative Agent in Pneumonia of Calves

Three field strains of Mycoplasma dispar were inoculated, by aerosol inhalation, into a total of eight naturally-born, colostrum-deprived calves. All three strains produced macroscopic pneumonia, each in one calf. Histopaithologically an exudative bronchitis accompanied by moderate interstitial cell proliferations was found. Reisolation studies indicated that the entire respiratory tract is the natural habitat of Mycoplasma dispar, which apparently does not spread via the blood stream.     

Extreme Levels of Noise Constitute a Key Neuromuscular Deficit in the Elderly

Fluctuations during isometric force production tasks occur due to the inability of musculature to generate purely constant submaximal forces and are considered to be an estimation of neuromuscular noise. The human sensori-motor system regulates complex interactions between multiple afferent and efferent systems, which results in variability during functional task performance. Since muscles are the only active component of the motor system, it therefore seems reasonable that neuromuscular noise plays a key role in governing variability during both standing and walking. Seventy elderly women (including 34 fallers) performed multiple repetitions of isometric force production, quiet standing and walking tasks. No relationship between neuromuscular noise and functional task performance was observed in either the faller or the non-faller cohorts. When classified into groups with either nominal (group NOM, 25^th –75^th percentile) or extreme (either too high or too low, group EXT) levels of neuromuscular noise, group NOM demonstrated a clear association (r²>0.23, p<0.05) between neuromuscular noise and variability during task performance. On the other hand, group EXT demonstrated no such relationship, but also tended to walk slower, and had lower stride lengths, as well as lower isometric strength. These results suggest that neuromuscular noise is related to the quality of both static and dynamic functional task performance, but also that extreme levels of neuromuscular noise constitute a key neuromuscular deficit in the elderly.